Plasminogen Activator Inhibitor type-1 (PAI-1) is the main inhibitor of tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA), the key serine proteases responsible for plasmin generation. PAI-1 regulates fibrinolysis by inhibiting plasminogen activation in the vascular compartment. Fibrinolysis is a tightly coordinated process for degrading fibrin clots formed by activation of the coagulation cascade. Dysregulation of the coagulation/fibrinolysis balance leads to abnormal haemostasis events like bleeding or thrombotic diseases. PAI-1 is also a key regulator of plasminogen activation in the pericellular compartment (intravascular and tissular) where receptor bound plasminogen is activated mainly by urokinase bound to the urokinase receptor (uPAR). By inhibiting pericellular proteolysis, PAI-1 regulates numerous cellular functions like extracellular matrix (ECM) degradation, growth factors activation and release from ECM, matrix metalloproteinases (MMP) activation and cellular apoptosis. Recently, protease-independent effects of PAI-1 have been identified through its interaction with cofactors (like vitronectin, heparin, glycosaminoglycan), uPAR-urokinase complexes or cellular receptors (LRP: low-density Lipoprotein Receptor-related Protein) or integrins affecting cell functions like adhesion/de-adhesion, migration, proliferation and intracellular bioactivity. By these cellular mechanisms and anti-fibrinolytic effects, a pathogenic role of PAI-1 has been established in tumor growth and metastasis, fibrosis, acute myocardial infarction and metabolic disorders like atherosclerosis, obesity and diabetes.
The Human SERPINE1 (PAI-1) gene is localized to chromosome 7, consists of eight introns and nine exons, and has a size of 12,169 b (Klinger, K. W. et al. Proc. Natl. Acad. Sci. USA 84:8548, 1987). PAI-1 is a single chain glycoprotein of approximately 50 kDa (379 amino acids) from the SERPIN (serine protease inhibitor) superfamily that is synthesized in the active conformation but spontaneously becomes latent in the absence of vitronectin (Vn). Vitronectin, the main cofactor of PAI-1, stabilizes the active conformation with the Reactive Center Loop (RCL) which is approximately 20 amino acids that are exposed on the surface. The mechanism of inhibition of PAI-1's two main targets (tPA and uPA) is a suicide inhibition. The RCL region of PAI-1 bears the bait peptide bond (R346-M347, also called P1-P′1), which bears the cleavage site for this serine protease. A Michaelis complex with tPA or uPA forms first, then the catalytic triad reacts with the bait peptide bond to form an acyl-enzyme complex that, after cleavage of the P1-P′1 peptide bond, induces strong conformation changes. The conformational changes cause insertion of the cleaved RCL into a β-strand with the protease staying covalently bound as an acyl enzyme with PAI-1. Under non-physiological circumstances, hydrolysis of this acyl-enzyme complex may induce release of the cleaved PAI-1 and free active protease (Blouse et al., Biochemistry, 48:1723, 2009).
PAI-1 circulates in blood at highly variable levels (nM range) and in excess over t-PA or uPA concentrations. PAI-1 exhibits structural flexibility and can be found in one of three conformations: (1) a latent conformation, (2) an active conformation, or (3) a substrate conformation (see FIG. 1). PAI-1 is mainly found as a noncovalent complex with vitronectin (Kd˜1 nM) that decreases latency transition by 1.5 to 3 fold. Latent, cleaved or complexed PAI-1 affinity for vitronectin is significantly reduced. Matrix bound vitronectin also localizes with PAI-1 in the pericellular space. Endothelial cells, monocytes, macrophages and vascular smooth muscle cells synthesize this PAI-1 which then can be stored in large amounts under latent form by platelets (in the α granule). PAI-1 is a fast and specific inhibitor (with the second order rate constant of 106 to 107 M−1s−1) of tPA and uPA in solution, but inactive against protease bound either to fibrin or their cellular receptors. Other proteases like thrombin, plasmin, activated Protein C cart be also inhibited by PAI-1 but less efficiently.
Several 3D structures of human PAI-1 have been solved since the first one described in 1992 (Mottonen et al., Nature 355:270, 1992) in the latent conformation. These 3D structures include mutant forms of PAI-1 in the substrate (Aertgeerts et al., Proteins 23:118, 1995), stabilized active conformation (Sharp et al., Structure 7:111, 1999), PAI complexed to Vitronectin-somatomedin B domain (Zhou et al., Nat. Struct. Biol. 10:541, 2003) or with inhibiting pentapeptide from the RCL loop (Xue et al., Structure 6:627, 1998). More recently, mouse PAI-1 structure in latent conformation was elucidated by Dewilde et al. (J. Struct. Biol. 171:95, 2010) and revealed differences with human PAI-1 in the RCL position, gate region and position of α-helix A. Structure/function relationships in PAI-1 have been studied by using more than 600 mutant proteins (reviewed by De Taeye et al., Thromb. Haemost. 92:898, 2004) to localize domains involved in the various activities of this multifunctional serpin.
Since PAI-1 can be synthesized by almost every cell type including hepatocyte, adipocyte, mesangial cell, fibroblast, myofibroblast, and epithelial cell, its expression greatly varies under physiological (e.g., circadian variation of plasma PAI-1 level) and pathological conditions (e.g., obesity, metabolic syndrome, insulin resistance, infection, inflammatory diseases, cancer). PAI-1 is considered to be an acute phase protein. Transcriptional regulation of PAI-1 mRNA expression is induced by several cytokines and growth factors (e.g., TGFβ, TNFα, EGF, FGF, Insulin, angiotensin II & IV), hormones (e.g., aldosterone, glucocorticoids, PMA, high glucose) and stress factors (e.g., hypoxia, reactive oxygen species, lipopolysaccharides).
Moreover, a polymorphism in the promoter (position-675) of the PAI-1 gene affects expression level. The 4G allele increases PAI-1 level and the 4G/4G variant (occurring in around 25% of the population) induces an increase of approximately 25% of plasma PAI-1 level in comparison to 5G/5G (25% occurrence and 4G/5G 50% occurrence). The 4G/4G polymorphism has been linked to myocardial infarction (Dawson et al., Arterioscler Thromb. 11:183, 1991), a specific type of pulmonary fibrosis (idiopathic interstitial pneumonia) (Kim et al., Mol Med. 9:52, 2003) and the 4G/4G genotype donor group is an independent risk factor for kidney graft loss due to Interstitial Fibrosis & Tubular Atrophy (Rerolle et al., Nephrol. Dial. Transplant 23:3325, 2008).
Several pathogenic roles have been attributed to PAI-1 in thrombotic diseases such as arterial and venous thrombosis, acute myocardial infarction, and atherosclerosis. Its involvement in metabolic disorders like insulin resistance syndrome and obesity is well recognized. PAI-1 is also known as a profibrotic factor for several organs and has been shown to be over-expressed in fibrotic tissues (i.e., liver, lung, kidney, heart, abdominal adhesions, skin: scar or scleroderma) (reviewed by Ghosh and Vaughan, J. Cell Physiol. 227:493, 2012). PAI-1 knock-out (KO) mice are protected from fibrosis in different models, such as liver (bile duct ligation or xenobiotic), kidney (unilateral ureteral obstruction model (UUO)), lung (bleomycin inhalation) (Bauman et al., J. Clin. Invest. 120:1950, 2010; Hattori et al., Am. J. Pathol. 164:1091, 2004; Chuang-Tsai et al., Am. J. Pathol.163:445, 2003) whereas in heart this deletion is protected from induced fibrosis (Takeshita et al., AM. J. Pathol. 164:449, 2004) but prone to age-dependent cardiac selective fibrosis (Moriwaki et al., Cric. Res. 95:637, 2004). Down-regulation of PAI-1 expression by siRNA (Senoo et al., Thorax 65:334, 2010) or inhibition by chemical compounds (Izuhara et al., Arterioscler. Thromb. Vasc. Biol. 28:672, 2008; Huang et al., Am. J. Respir. Cell Mol. Biol. 46:87, 2012) have been reported to decrease lung fibrosis whereas PAI-1 overexpression of wild type (Eitzman et al., J. Clin. Invest. 97:232, 1996) or a PAI-1 mutant retaining only vitronectin binding but not tPA inhibitor function exacerbates lung fibrosis (Courey et al., Blood 118:2313, 2011).
Bile duct ligation (BDL) liver fibrosis is attenuated by antibody neutralizing PAI-1 (U.S. Pat. No. 7,771,720) whereas down-regulation by siRNA attenuates BDL and xenobiotic induced liver fibrosis (Hu et al., J. Hepatol. 51:102, 2009). PAI-1 KO mice were protected from cholestatic-induced liver damage and fibrosis in BDL (Bergheim et al., J. Pharmacol. Exp. Ther. 316:592, 2006; Wang et al., FEBS Lett. 581:3098, 2007; Wang et al., Hepatology 42:1099, 2005) and from angiotensin II induced liver fibrosis (Beier et al., Arch. Bioch. Biophys. 510:19, 2011).
PAI-1 KO mice are protected from renal fibrosis in the UUO model (Oda et al., Kidney Int. 60, 587, 2001), in diabetic nephropathy (Nicholas et al., Kidney Int. 67:1297, 2005) and in angiotensin II induced nephropathy (Knier et al., J. Hypertens. 29:1602, 2011; for reviews see Ma et al. Frontiers Biosci. 14:2028, 2009 and Eddy A. A. Thromb. Haemost. 101:656, 2009). In contrast, PAI-1 over expressing mice display more severe fibrosis and increased macrophage recruitment following UUO (Matsuo et al., Kidney Int. 67:2221, 2005; Bergheim et al., J. Pharmacol. Exp. Ther. 316:592, 2006). Non-inhibitory PAI-1 mutant (PAI-1 R) has been shown to protect mice from the development of fibrosis in experimental glomerulonephritis (thy1) in rat by decreasing urinary protein expression and glomerular matrix accumulation (Huang et al., Kidney Int. 70:515, 2006). Peptides blocking PAI-1 inhibit collagen 3, 4 and fibronectin accumulation in UUO mice (Gonzalez et al., Exp. Biol. Med. 234:1511, 2009).
PAI-1, as a target for numerous pathologies, has been the focus of intensive research to inhibit its activity or to regulate its expression for the last 20 years. Chemical compounds (Suzuki et al., Expert Opin. Investig. Drugs 20:255, 2011), monoclonal antibodies (Gils and Declerk, Thromb Haemost; 91:425, 2004), peptides, mutants (Cale and Lawrence, Curr. Drug Targets 8:971, 2007), siRNA or anti-sense RNA have been designed to inhibit its various function or to regulate its expression. However, despite the intensive research, the problem of developing a therapeutically effective modulator of PAI-1 still remains to be solved. Accordingly, there is a need in the art for novel agents that inhibit the PAI-1 activity for use in the treatment of PAI-1-mediated human pathologies.